JP2004531578A - Intradermal delivery of vaccines and gene therapeutics by microcannula - Google Patents

Abstract

Methods and devices for administering vaccines and gene therapy agents to the intradermal layer of the skin.

Description

【Technical field】
[0001]
The present invention relates to a method and a device for administering a vaccine and a gene therapy agent to the intradermal layer of the skin.
[Background Art]
[0002]
The importance of effective and safe administration of drug substances for prophylactic, diagnostic or therapeutic purposes has been recognized for many years. The use of conventional needles has provided one approach for delivering drug substances by administration through the skin to humans and animals over time. Considerable efforts have been made to achieve reproducible and effective delivery through the skin while improving ease of injection and reducing patient anxiety and / or pain associated with conventional needles. In addition, some delivery systems do not use a needle at all and rely on iontophoretic currents or chemical mediators or external propulsion, such as electroporation or thermal poration or sonication, to reap the skin. Breaks the outer stratum corneum and delivers the substance through the surface of the skin. However, such delivery systems are reproducible, do not break the skin barrier, or do not deliver the drug substance to a certain depth below the surface of the skin, and the clinical outcome may be undefined. Thus, mechanically breaking the stratum corneum, such as with a needle, would provide the most reproducible method of administration of a substance through the surface of the skin, and the placement of the administered substance would be controlled and reliable. Conceivable.
[0003]
Methods of delivering substances below the surface of the skin include transdermal administration almost exclusively, ie, delivery of the substance through the skin to the lower skin. Transdermal administration includes subcutaneous, intramuscular or intravenous routes of administration, of which intramuscular (IM) and subcutaneous (SC) injections have been most commonly used.
[0004]
Anatomically, the outer surface of the body consists of two major layers of tissue that together make up the skin: the outer epidermis and the underlying dermis (for a review, see Nonpatent Literature). 1). The epidermis is divided into five layers with a total thickness of 75-150 μm. Below the epidermis is the dermis, which contains two layers, the outer part is called the papillary dermis and the deeper layer is called the reticular dermis. The dermal papilla contains many microcirculating blood and lymph plexus. In contrast, the reticular dermis is relatively acellular and avascular, and is composed of dense collagen tissue and elastic connective tissue. Underneath the epidermis and dermis is the subcutaneous tissue, also called hypodermis, consisting of connective and adipose tissue. The muscle tissue is below the subcutaneous tissue.
[0005]
As noted above, both subcutaneous and muscular tissues have generally been used as sites of administration for drug substances. However, the dermis has rarely been targeted as a site of administration of the substance, at least in part because it is difficult to accurately position the needle in the intradermal space. Furthermore, even in the dermis, in particular, the dermal papilla is known to have a high degree of vascularity, and in order to obtain an improved absorption profile of the administered substance compared to subcutaneous administration, this high degree The use of vascularity has not been previously recognized. This is because small drug molecules are much easier to target than the dermis, and are generally rapidly absorbed after administration to the subcutaneous tissue, which could be expected. On the other hand, macromolecules, such as proteins, are generally not well absorbed through the capillary epithelium, regardless of the degree of vascular distribution, so macromolecules are also more difficult to achieve intradermally because Would not have been expected to achieve a significant absorption advantage over subcutaneous administration.
[0006]
One method of administration to the area below the skin surface and in the intradermal space is routinely used in the Mantouts Berglin test. In this method, the purified protein derivative is injected at a shallow angle to the skin surface using a 27 or 30 gauge needle and a standard syringe (Non-Patent Document 2). The Mantoux technique involves inserting the needle into the skin from the side, and then "propelling" the needle further into the intradermal tissue. This approach is known to be quite difficult to implement and requires special training. Depending on the degree of inaccuracy of the injection location, a significant number of false negative test results are obtained. In addition, this test required a local injection to elicit an injection site response, and the Mantoux method did not lead to the use of intradermal injections for systemic administration of the substance.
[0007]
Another group reported what was described as an intradermal drug delivery device (U.S. Patent No. 6,049,059). Injection has been shown to be slow, and the injection site was intended to be some area under the epidermis, ie, at the interface between the epidermis and the dermis or inside or under the subcutaneous tissue of the dermis. However, this reference does not provide any teachings suggesting selective administration into the dermis, nor does it suggest that vaccines or gene therapy agents can be delivered in this manner.
[0008]
To date, a number of therapeutic proteins and low molecular weight compounds have been delivered intradermally and have been used to effectively elicit pharmacologically useful responses. Most of these compounds (eg, insulin, Neupogen, hGH, calcitonin) are hormonal proteins, not engineered receptors or antibodies. To date, all administered proteins have shown a faster onset of uptake and distribution (vs. SC) and some effects associated with ID administration, including in some cases increased bioavailability. Was.
[0009]
Skin tissue represents an attractive target site for the delivery of vaccines and gene therapy agents. In the case of vaccines (gene and conventional), the skin is an attractive target because high concentrations of antigen presenting cells (APCs) and APC precursors are found in this tissue, especially in epidermal Langerhans cells and dermal dendritic cells Part. Several gene therapy agents have been designed for the treatment of skin disorders, skin diseases and skin cancers. In such cases, it is desirable to deliver the therapeutic agent directly to the affected skin tissue. Furthermore, skin cells are attractive targets for gene therapy agents whose encoded protein or proteins are active at sites remote from the skin. In such cases, skin cells (eg, keratinocytes) function as “bioreactors” that produce therapeutic proteins that can be rapidly absorbed into the systemic circulation through the dermal papilla. In other cases, it may be desirable to have the vaccine or therapeutic agent have direct access to the systemic circulation for treatment of disorders at sites remote from the skin. In such a case, distribution to the whole body can be realized via the papillary dermis.
[0010]
However, as noted above, intradermal (ID) injection using a standard needle and syringe is technically very difficult to perform and painful. The prior art includes several references on ID delivery of DNA-based and conventional vaccines and therapeutics, at least in part because existing technologies have difficulty accurately targeting ID tissues. , The results were not consistent.
[0011]
Virtually all human vaccines currently on the market are administered by the IM or SC route. Of the 32 vaccines manufactured in 2001 by four major global vaccine manufacturers (Aventis-Pasteur, GLaxoSmithKline, Merck, Wyeth), only two were approved for ID (2001 Pharmaceutical Drugs). Handbook). In fact, the package insert of six of these 32 vaccines states that the ID route is not used. Various published pre- and early-stage clinical trials compare ID delivery by eliciting a stronger immune response than IM or SC injection, or at lower doses compared to the doses given with IM or SC This is despite the suggestion of improving the vaccine by eliciting an immune response (Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5, Non-Patent Document 6, Non-patent document 7, Non-patent document 8, Non-patent document 9). In some cases, improved efficacy of the vaccine after ID delivery was observed, while in other cases such benefits were not observed (Non-Patent Document 10, Non-Patent Document 11, Non-Patent Document 11). Patent Document 12, Non-patent Document 13, Non-patent Document 14).
[0012]
A major factor that has prevented the widespread use of ID delivery routes and has led to the above conflicting results is the lack of suitable equipment to achieve reproducible delivery to the epidermal and dermal layers. Standard needles commonly used to inject vaccines are too large to accurately target these tissues when inserted into the skin. The most common method of delivery is via a Mantoux injection using a standard needle and injection. This method is difficult to perform, unreliable, and painful to the subject. Therefore, there is a need for devices and methods that allow for efficient, accurate and reproducible delivery of vaccines and gene therapy agents to the intradermal layer of the skin.
[0013]
[Patent Document 1]
U.S. Pat. No. 5,997,501
[Patent Document 2]
EP-A-1092444 A1
[Patent Document 3]
US Patent Application No. 606909 filed on June 29, 2000
[Patent Document 4]
U.S. Patent Application No. 4,177,671, filed October 14, 1999
[Patent Document 5]
U.S. Pat. No. 5,589,466
[Patent Document 6]
U.S. Pat. No. 5,593,972
[Patent Document 7]
U.S. Pat. No. 5,703,055
[Patent Document 8]
U.S. patent application Ser. No. 10 / 142,966 filed May 13, 2002.
[Patent Document 9]
U.S. Pat.
[Patent Document 10]
EP-A-1092444
[Non-patent document 1]
Physiology, Biochemistry, and Molecular Biology of the Skin, Second Edition, LA Goldsmith, Ed., Oxford University Press, New York, 1991
[Non-patent document 2]
Flynn et al, Chest 106: 1463-5, 1994
[Non-Patent Document 3]
Playford, EG et al, Infect, Control Hosp. Epidemiol. 23: 87, 2002
[Non-patent document 4]
Kerr, C. Trends Microbiol, 9: 415, 2001
[Non-Patent Document 5]
Rahman, F. et al., Hepatology 31: 521, 2000
[Non-Patent Document 6]
Carlsson, U. et al., Scan J. Infect. Dis. 28: 435 1996
[Non-Patent Document 7]
Propst, T. et al., Amer. J. Kidney Dis. 32: 1041, 1998.
[Non-Patent Document 8]
Nagafuchi, S. et al., Rev Med Virol, 8: 97, 1998.
[Non-Patent Document 9]
Henderson, EA, et al., Infect.Control Hosp Epidemiol. 21: 264, 2000
[Non-Patent Document 10]
Crowe, Am. J. Med.Tech. 31: 387-396, 1965
[Non-Patent Document 11]
Letter to British Medical Journal 29/10/77, p.1152
[Non-Patent Document 12]
Brown et al., J. Infect. Dis. 136: 466-471, 1977.
[Non-patent document 13]
Herbert & Larke, J. Infect. Dis. 140: 234-238, 1979
[Non-patent document 14]
Ropac et al. Periodicum Biologorum 103: 39-43, 2001
[Non-Patent Document 15]
Immunology Today, Apr 21 (4): 163-165, 2000
DISCLOSURE OF THE INVENTION
[Problems to be solved by the invention]
[0014]
The present invention improves the clinical utility of ID delivery of vaccines and gene therapy agents to humans or animals. The method uses a device to target the intradermal space directly and deliver the substance to the intradermal space as a bolus or by injection. It has been discovered that placing the substance in the dermis results in an effective and / or improved response to vaccines and gene therapy agents. The device is designed to prevent leakage of material from the skin and improve absorption or cellular uptake within the intradermal space. The immune response to vaccine delivery according to the method of the present invention has been found to be equal to or better than normal IM delivery of the vaccine, and ID administration according to the method of the present invention has the additional advantages of ID delivery. Have been found to often lead to improved clinical results.
[0015]
The present invention also relates to a method and a device for delivering a vaccine or genetic material to an individual based on direct targeting of the skin space, such a method for improving and / or improving the delivery of the vaccine or genetic material. The response can be improved. Vaccine and gene therapy by using direct intradermal (ID) administration means (hereinafter referred to as dermal access means), eg, injection and infusion systems with microneedles, or other methods of accurately targeting the intradermal space The effectiveness of many substances, including agents, can be improved over traditional parenteral routes of administration, such as subcutaneous and intramuscular delivery.
[0016]
Accordingly, it is an object of the present invention to provide a method for accurately targeting and delivering a vaccine or medicament containing genetic material to an ID tissue to produce an immune and therapeutic response.
[0017]
It is a further object of the present invention to provide a method of improving systemic immunity or therapeutic response to pharmaceuticals containing vaccines (conventional or genetic) or genetic material by accurately targeting ID tissues. .
[0018]
Provided is a method for improving the availability of vaccines (conventional or genetic) against APCs present on the skin by precisely targeting ID tissues to enable an antigen-specific immune response to the vaccine It is another object of the present invention to do so. This often allows smaller amounts of the substance to be administered by the ID route.
[0019]
It is another object of the present invention to provide a method for improving the delivery of pharmaceuticals containing genetic material for the treatment of skin diseases, genetic skin disorders or skin cancers by accurately targeting ID tissues. The resulting genetic material is then expressed by cells in the targeted ID tissue.
[0020]
It is another object of the present invention to provide a method for improving the delivery of pharmaceuticals containing genetic material for the treatment of diseases, genetic disorders or cancers affecting tissues away from the skin by accurately targeting ID tissues. Is the purpose. The resulting genetic material is then expressed by cells within the targeted ID tissue, tissues away from it, or both.
[Means for Solving the Problems]
[0021]
These and other advantages of the present invention are realized by directly targeting the delivery of a substance to a preferred depth for a particular therapeutic or prophylactic agent. We can unexpectedly improve the response to vaccines and gene therapeutics by clearly targeting the delivery of substances to the intradermal space, and in many situations, together with the clinical benefits obtained. I found that it can be changed.
BEST MODE FOR CARRYING OUT THE INVENTION
[0022]
As used herein, "intradermal" (ID) refers to the ability of a substance to be rapidly absorbed systemically or to a vaccine (conventional or genetic) or a gene therapy agent. In this case, it means that the substance is administered intradermally in such a way that the substance easily reaches the abundantly vascularized dermal papilla, which is directly absorbed by the cells of the skin. In the case of a genetic vaccine, the intended target cells include APCs (including epidermal Langerhans cells and dermal dendritic cells). In the case of gene therapy for diseases, genetic disorders or cancers affecting tissues away from the skin, the intended target cells include keratinocytes or other skin cells capable of expressing a therapeutic protein. In the case of a gene therapy for a disease, genetic disorder or cancer affecting the skin, the intended target cells include skin cells affected by the disease, genetic disorder or cancer.
[0023]
As used herein, "targeted delivery" refers to the delivery of a substance to a targeted depth, resulting in the same response in the individual being treated, but with another accepted means of delivery (e.g., (Including topical, subcutaneous or intramuscular), including delivery with less pain, greater reproducibility, or other benefits.
[0024]
As used herein, "improved response" refers to a response equivalent to a reduced amount of the compound administered, or an increased response to the same amount of the compound administered by another means of delivery. Or other therapeutic or immunological benefits.
[0025]
The term “needle” or “needles” as used herein is intended to include all such needle-like structures. The term microcannula or microneedle, as used herein, is intended to include structures of about 31 gauge, generally less than 31-50 gauge, when such structures are cylindrical. It is. Non-cylindrical structures included in the term microneedles are of equivalent diameter and include pyramids, rectangles, octagons, wedges and other geometric shapes.
[0026]
As used herein, the term "bolus" means an amount delivered in less than 10 minutes. "Rapid bolus" means an amount delivered in less than a minute. "Infusion" means the delivery of a substance over a period of more than 10 minutes.
[0027]
The term "nucleic acid" includes a polynucleotide, RNA, DNA or RNA / DNA hybrid sequence of a plurality of nucleotides in single- or double-stranded form and can be formulated and delivered using the methods of the invention. It can be of any size possible. The nucleic acid may be in "antisense" form. “Nucleic acid-derived entity” means that the nucleic acid is wholly or partially composed of a nucleic acid.
[0028]
"Gene therapy agent" means an agent that is intended to be delivered to or taken up by cells of an individual to be treated for the uptake and expression of genetic material. The gene therapy agent is usually a polynucleotide encoding the peptide, polypeptide, protein or glycoprotein of interest, optionally contained in a vector or plasmid that is readily available for any additional nucleic acid sequences required for expression. including.
[0029]
When referring to the administration of a vaccine or gene therapy agent, the term "simultaneously" means two administrations within the same 24 hour period, whereas "sequentially" or "sequentially" means that the administration period is 24 hours. This means that they are farther apart. Simultaneous administration refers to administration that occurs at the same medical visit, but may be sequential or sequential, depending on the effect of the particular vaccine or gene therapy, for days, weeks, months, or even a few months. One skilled in the art will recognize that referring to administration that is spaced annually. In one preferred embodiment, "sequential" or "consecutive" refers to administrations that are one to six weeks apart.
[0030]
The desired treatment or immune response is directly related to the ID target depth. These results can be obtained by placing the substance in the upper region of the dermis, i.e., the papillary dermis, or the upper part of the reticulated dermis, where there are relatively few blood vessels where the substance readily diffuses into the papillary dermis. Preferred is placement of the material primarily at a depth of at least about 0.025 mm to about 2.5 mm.
[0031]
In particular, for vaccines, it is preferred that delivery be performed just below the stratum corneum and at a target depth that includes the epidermis and upper dermis (about 0.025 mm to about 2.5 mm). In the case of therapeutic agents targeting skin cells, the preferred target depth depends on the particular cell targeted. For example, for therapeutic agents targeting Langerhans cells, delivery will typically need to include, at least in part, epidermal tissue depth in the range of about 0.025 mm to about 0.2 mm in humans. . For therapeutic agents requiring systemic circulation, a preferred target depth is at least about 0.4 mm, most preferably at least about 0.5 mm to about 2.5 mm or less, more preferably about 2.0 mm or less, most preferably Up to about 1.7 mm or less, resulting in delivery of the substance to the desired dermal layer. The placement of the substance primarily at a greater depth and / or in the lower reticular dermis is generally considered less desirable.
[0032]
The dermal access means used for ID administration according to the present invention is not critical as long as it provides the depth of insertion into the subject's skin necessary to obtain the target delivery depth of the substance. In most cases, the device will penetrate the skin to a depth of about 0.5-2 mm. The dermal access means may include all known types of conventional injection needles, catheters, microcannulas or microneedles used alone or in multiple needle arrangements.
[0033]
By varying the target depth of delivery of the substance by the dermal access means, the behavior of the drug or substance can be adapted to the desired clinical application most appropriate to the individual patient's condition. The target depth of delivery of the substance by the dermal access means can be adjusted manually by an expert, with or without the aid of indicator means to indicate when the desired depth has been reached. However, it is preferred that the device has means for regulating skin penetration to a desired depth within the intradermal space. This is generally achieved by an enlarged portion or hub associated with the dermis access means which may take the form of a backing structure or platform to which the needle is attached. The length of the microneedles as the dermal access means can be easily changed during the manufacturing process, and the microneedles manufactured on a daily basis also reduce the pain or other sensations during injection or infusion. Very sharp and very small gauge size. They can be used in the present invention as individual single lumen microneedles, or multiple microneedles can be used to increase the rate of delivery, i.e., the amount of material delivered within a given time period A linear array or a two-dimensional array can be used. Microneedles having one or more side holes are also included as dermis access means. Microneedles can be incorporated into various devices such as holders and housings that also serve to limit the depth of penetration. The dermis access means of the present invention may include a reservoir or a pump or other means for delivering a drug or other substance under pressure containing the substance prior to delivery. Alternatively, such additional components may be external to the device containing the dermis access means. The dermal access means may include passive or active safety features to prevent or reduce accidental damage.
[0034]
In one embodiment of the invention, ID injections can be performed reproducibly using one or more fine gauge microcannulas inserted perpendicular to the skin surface. This method of delivery ("microdermal delivery" or "MDD") is easier to implement than standard Mantoux injection, and has a limited and controlled depth of penetration into the skin, resulting in an invasive And less painful. In addition, the present invention allows a similar or greater biological response as measured by gene expression and immune response compared to a standard needle to be obtained using an MDD device. The optimal dosage depth for a given substance in a given animal species can be determined by one of ordinary skill in the art without undue experimentation.
[0035]
Most preferred are delivery devices that allow the dermal access means to be placed at the appropriate depth within the skin, control the rate and rate of liquid delivery, and accurately deliver the substance to the desired location without leaking. Methodologies and devices using microcannulas and microneedles are described in US Pat. Standard steel cannulas can also be used for intradermal delivery using the devices and methods described in U.S. Patent No. 6,037,064, the contents of each of which are incorporated herein by reference. These methods and devices include finer gauge (about 30 G) "microcannulae" with penetration depth limitations defined by the total length of the cannula exposed beyond the equipment that limits the overall length or depth of the cannula. And the like, delivery of a substance. Although these methods and devices are for the delivery of substances via a 30 or 31 gauge cannula, the present invention also provides a 34G or thinner "microcannula" which includes, if desired, a limitation or control of the depth of the penetrating means. Used. Targeted delivery of a substance can be via a single microcannula or an array of microcannulas (or "microneedles"), for example, including or attached to a reservoir containing the substance to be administered. It is within the scope of the present invention that this can be achieved via three to six microneedles mounted on the injection device.
[0036]
Using the methods of the present invention, vaccines and gene therapy agents can be administered as a bolus or by infusion. It is understood that the bolus administration or delivery can be effected by means of a rate controlling means, such as a pump, or without specific rate controlling means, such as by self-injection of the user. The above advantages are best recognized by the targeted and accurate direct delivery of the substance to the skin tissue compartment, including the epidermis. This is achieved, for example, with a microneedle system having an outer diameter of less than about 250 microns and an exposed length of less than 2 mm. By "exposed length" is meant the length of a thin hollow cannula or needle available to penetrate the patient's skin. Such systems use known methods for a variety of materials such as steel, silicon, ceramics and other metals, plastics, polymers, sugars, biological and / or biodegradable materials and / or combinations thereof. Can be built.
[0037]
It has been found that certain features of the intradermal administration method produce the most effective results. For example, it has been found that placing the needle outlet in the skin significantly affects the clinical response to vaccine or gene therapy delivery. The outlet of a conventional or standard gauge needle having a bevel angle cut at an angle of 15 degrees or less has a relatively large "exposed height". As used herein, the term exposed height refers to the length of the axial opening of the cannula due to the oblique cut. When the standard needles are placed at the desired depth in the intradermal space (at about 90 degrees relative to the skin), the back exposure exerted by the skin itself, or injection or The substance normally flows out of the skin due to the increased pressure due to the liquid accumulating on injection. Generally, the exposed height of the present invention is from 0 to about 1 mm. The outlet of the needle having an exposed height of 0 mm has no cut in the slope (ie, a 90 degree tilt angle), and the outlet is at the tip of the needle. In this case, the depth of the outlet is the same as the depth of penetration of the needle. The outlet of the needle formed by the bevel cut or the opening in the side of the needle has a measurable exposed height. For a beveled needle, the exposed height of the needle outlet is determined by the diameter of the needle and the angle of the primary bevel cut ("tilt angle"). Generally, a tilt angle of more than 20 ° is preferred, more preferably between 25 ° and 40 °. It is understood that a single needle may have multiple openings or outlets suitable for delivering a substance to the skin space.
[0038]
Thus, the exposed height, and in the case of a cannula having an opening on the side, its position along the axis of the cannula contributes to the depth and specificity with which the substance is delivered. Other factors considered in the cannula alone or in combination with the cannula, such as delivery rate and total volume of liquid delivered, contribute to the targeted delivery of the substance and vary such parameters to optimize the result That is within the scope of the present invention.
[0039]
It has also been found that by controlling the injection or infusion pressure, high back pressure during ID administration can be avoided. By applying a constant pressure directly to the liquid interface, a more constant delivery rate can be achieved that can optimize absorption and obtain an improved response to the delivered vaccine or therapeutic agent dose. Delivery rate and volume can also be controlled to prevent wheals at the delivery site and to prevent back pressure from pushing the dermal access means out of the skin. Appropriate delivery rates and volumes to achieve these effects for a selected agent can be determined empirically using routine techniques, without undue experimentation. Increasing the spacing between the needles allows for a wider liquid distribution and increased delivery rate or a larger liquid volume.
[0040]
In one embodiment, to deliver a substance, a dermis access means is placed adjacent to the skin of the subject to provide targeted access directly into the intradermal space, allowing one or more substances to be delivered to the subject. Is delivered or administered to the intradermal space where it can act locally or be absorbed by the bloodstream and distributed throughout the body. In other embodiments, the dermal access means is placed substantially perpendicular to the skin surface and one or more cannulas are inserted vertically. The dermal access means can be connected to a reservoir containing the substance or substances to be delivered. The form of the substance or substances to be delivered or administered may be in the form of solutions, emulsions, suspensions, gels, suspended or dispersed microparticles and nanoparticles in a pharmaceutically acceptable diluent or solvent. Particles as well as the in situ forming vehicles described above. Delivery from the reservoir to the intradermal space may be active passively without applying external pressure or other drive means and / or active by applying pressure or other drive means to the substance or substances to be delivered. Can happen. Examples of preferred pressure generating means include pumps, syringes, elastic membranes, gas pressures, piezoelectric, electromotive, electromagnetic pumping, helical or flat springs or washers or washers or combinations thereof. If desired, the rate of delivery of the substance can be variously controlled by the pressure generating means. As a result, the substance enters the intradermal space and is absorbed in an amount and at a rate sufficient to obtain a clinically effective result.
[0041]
Materials that can be delivered by the methods of the present invention include vaccines with or without carriers, adjuvants and vehicles, including prophylactic and therapeutic antigens, including: But not limited to: subunit proteins, peptides and polysaccharides, polysaccharide complexes, toxoids, gene-based vaccines, live attenuated bacteria or viruses, mutant bacteria or viruses, reconstructed bacteria or viruses, inactivated bacteria or viruses Whole cells or components thereof (eg, mammalian cells), cellular vaccines (eg, autologous dendritic cells) or components thereof (eg, exosomes, dexosomes, membrane fragments or vesicles), live viruses, live bacteria, Adenoviruses, retroviruses, alphaviruses, flaviu Viral and fungal vectors, including those derived from the Rus and Vaccinia viruses: addiction (eg, cocaine addiction), malignant pustules, arthritis, cholera, diphtheria, dengue, tetanus, lupus, multiple sclerosis, parasitosis, psoriasis , Lyme disease, meningococcal disease, measles, epidemic parotitis, rubella, blister, yellow fever, respiratory syncytial virus, tick-borne Japanese encephalitis, pneumococcus, smallpox, streptococcus, staphylococcus, typhoid, influenza Including hepatitis A, B, C and E, otitis media, rabies, polio, HIV, parainfluenza, rotavirus, Epstein-Barr virus, CMV, chlamydia, atypical hemophilus, hemophilus influenza B (HIB), Moraki Serracatalaris, human papillomavirus, tuberculosis including BCG, gonorrhea, asthma, atherosclerosis, Laria, E. coli, Alzheimer's disease, H. Pylori, Salmonella, diabetes, cancer, herpes simplex, human papilloma, Yersinia pestis, traveler's disease, West Nile encephalitis, Campylobacter (Campobacter), C. difficile. Suitable exemplary compositions for gene immunization are described, for example, in US Pat.
[0042]
Particularly preferred substances that can be induced by the method of the present invention include nucleic acids, nucleic acid-derived substances, and gene therapy agents used for the prevention, diagnosis, alleviation, treatment or treatment of diseases. Suitable adjuvants for inclusion in the vaccine are known to those skilled in the art. Additional substances for enhancing the immune response that can be used in the present invention are described in US Pat.
[0043]
Particularly preferred gene therapy agents include, but are not limited to, melanoma, cutaneous T-cell lymphoma, Kaposi's sarcoma, cutaneous squamous cell carcinoma and basal cell carcinoma, cancer, adenosine deaminase deficiency, hyperproliferation including but not limited to psoriasis Hereditary skin diseases, including, but not limited to, hereditary skin diseases, exfoliative keratosis, epidermolysis bullosa, lamellar ichthyosis and X-linked ichthyosis, growth disorders, human growth Includes those indicated for the treatment of hormone deficiency, including but not limited to hormone deficiency, atherosclerosis, transferrin deficiency, and gene therapy agents indicated for wound healing and tissue regeneration. Suitable exemplary compositions for suitable gene therapy agents are described, for example, in US Pat.
[0044]
The substance can be delivered into the skin in a pharmaceutically acceptable form. Vaccines that can be used in the methods of the invention may include adjuvants and carriers or vehicles appropriate to the particular formulation, as known to those skilled in the art.
[0045]
Pharmaceutically acceptable peptide and polypeptide formulations for the present invention, including formulations of allergen compositions, are also well known in the art. The nucleic acid used in the method of the present invention may be RNA or DNA or a combination thereof. They can be in any physical form suitable for ID administration and uptake and expression by cells. The DNA and / or RNA may be contained in a viral vector or liposome, or may be delivered as a free polynucleotide, such as a plasmid known in the art. Nucleic acids are generally formulated as pharmaceutically acceptable formulations, such as solutions, gels or suspensions that are compatible with the nucleic acids.
[0046]
Generally, the expert will use a syringe to remove the appropriate volume from a septum-sealed vial to administer the vaccine or other drug. The vaccine is then administered to the patient using the same syringe. However, microneedles or microcannulas, typically 0.1-2 mm in length, are somewhat inadequate in length to allow full penetration of the septum, as well as sufficient for subsequent use in patients. It is generally too fragile to puncture the septum of the vial to extract the drug while maintaining a sharp and straight shape. The use of such microdevices to puncture a septum may result in a plugged needle lumen. In addition, thin gauges, typically 31-50 gauge microcannulas, significantly reduce, for example, the volume that can be transferred from a needle to a syringe. This is inconvenient for most professionals accustomed to quickly transferring liquids from vials using conventional equipment, and will significantly increase the time they spend on patients. Additional factors to consider in the widespread use of microdevices include the formulation of most drugs and vaccines to accommodate the reduced total volume (10-100 μl) used or delivered by the microdevice. Includes the need for rework. Accordingly, it would be desirable to provide a kit that includes a device in combination with, or suitable for incorporation with, the substance to be delivered.
[0047]
Kits and the like containing the administration device and the therapeutic composition are well known in the art. However, the application of minimally invasive ID microdevices for the delivery of drugs and vaccines is safe, effective and consistent with respect to administering prescriptions to enable immune and therapeutic responses. It clearly illustrates the direct need for a formulation-device link to provide a means.
[0048]
The kit provided by the present invention has at least one hollow microneedle designed to deliver the substance intradermally to a depth of 0.025-2 mm, wherein the microneedle is a vaccine or gene therapy drug. A delivery device is provided in fluid communication with or arranged in fluid communication with a reservoir adapted to contain a dose. In a preferred embodiment, the kit also contains an effective amount of a vaccine or gene therapy agent, which is an integral part of the delivery device or optionally contained in a reservoir that can be operatively attached thereto. The hollow microneedles are preferably about 31-50 gauge, and may be part of an array of 3-6 microneedles, for example.
[0049]
In a particularly preferred embodiment, the kit of the present invention comprises a hub portion attachable to a pre-fillable reservoir for storing the vaccine;
At least one hollow microneedle supported by the hub portion and having a forward tip extending away from the hub portion;
A generally planar skin barrier that includes a limiter portion surrounding the microneedle and extending away from the hub portion toward the front end of the microneedle, the limiter extending in a plane generally perpendicular to the axis of the microneedle. And one or more anterior tips of said one or more microneedles, wherein said one or more microneedles are adapted to be received on the skin of a mammal for performing an intradermal injection of a vaccine. Extending over a distance of 0.5 to 2.0 mm, the limiter portion limits penetration of the microneedle into the dermis layer of mammalian skin.
[0050]
To use the kit as envisioned by the present invention, the expert will break the hermetic seal to allow access to the microdevice, and optionally the vaccine or immunogenic or therapeutic composition. Would. The composition can be pre-filled into the microdevice in any form, including but not limited to gels, pastes, oils, milks, particles, nanoparticles, microparticles, suspensions or liquids. The composition may be packaged separately in a kit package, such as a reservoir, vial, tube, blister, pouch, or the like. One or more components of the formulation may be lyophilized, dried, spray dried, or in any other reconstitutable form. If desired, various reconstitution media, cleaning or disinfecting or topical sterilizing agents (alcohol wipes, iodine) can be further provided. The professional will, if necessary, fill or incorporate the substance into the device and then administer the formulation to the patient using an ID injection microdevice.
[0051]
Having generally described the invention, various examples for practicing the invention will now be described with reference to the following specific, but non-limiting, examples and figures.
[0052]
A representative sample of a dermal access microdevice (MDD device) containing a single needle was prepared from 34 gauge steel stock (MicroGroup, Inc., Medway, Mass.) And a single 28 ° ramp was used with an 800 grit carborundum grinding wheel. And ground. The needles were cleaned by sequential sonication in acetone and distilled water and effluent was checked using distilled water. The microneedle was fixed to a small gauge catheter tube (Maersk Medical) using an ultraviolet curable epoxy resin. The length of the needle was set using a mechanical indexing plate, the hub of the catheter tube functioned as a depth limiting adjuster, and confirmed by light microscopy. The length of the exposed needle was adjusted to 1 mm using an index plate. The connection with the syringe was made via an integral Luer adapter at the entrance of the catheter. During the injection, the needle was inserted perpendicular to the skin surface and held in place by gentle gentle hand pushing for bolus delivery. The function of the device and the flow of liquid were checked immediately before and immediately after the injection. A 30/31 gauge intradermal needle device having a 1.5 mm exposure length controlled by a depth limiting hub as described in US Pat.
Embodiment 1
[0053]
Model Gene Therapy / Prophylactic Agent ID Delivery and Expression, Guinea Pig Model
Uptake and expression of DNA in vivo by cells is important for effective gene therapy and genetic immunization. Plasmid DNA encoding the reporter gene firefly luciferase was used as a model gene therapy (Aldevron, Fargo, ND). DNA was injected intradermally (ID) into Hartley guinea pigs (Charles River, Raleigh, NC) using a standard 30G needle by the Mantoux method (ID-Mantoux) or was inserted almost vertically and made of 34G steel with a length of 1 mm. ID was delivered by MDD using a microcannula (MDD device) (ID-MDD). Plasmid DNA was applied topically to shaved skin as a negative control (plasmid size too large to allow passive uptake into skin). The total dose was 100 μg per animal and was delivered as a rapid bolus injection (less than 1 minute) using a 1 cc syringe in a total dose volume of 40 μl PBS. A full thickness skin biopsy sample at the site of administration was collected 24 hours after delivery, homogenized, and further processed to measure luciferase activity using a commercially available assay (Promega, Madison, WI). Luciferase activity was normalized to total protein content in tissue samples as measured by the BCA assay (Pierce, Rockford, IL) and expressed as relative light units (RLU) per mg total protein (group for mantoux and negative controls). N = 3, n = 6 for MDD devices).
[0054]
The results (FIG. 1) show strong luciferase expression in both ID injection groups. Mean luciferase activity in the MDD and Mantoux groups was 240 and 220 times higher than the negative control, respectively. Luciferase expression levels in local controls were not significantly greater than untreated skin sites (data not shown). These results indicate that the method of the present invention using the MDD device is at least as effective as the Mantoux method for delivering genetic material to ID tissues, and that significant levels of localized genes in vivo by skin cells It shows that expression is brought about.
Embodiment 2
[0055]
Model Gene Therapeutic / Prophylactic Agent ID Delivery and Expression, Rat Model
To assess the utility of this platform across multiple animal species, experiments similar to those described in Example 1 above (without the Mantoux control) were performed in Brown-Norway rats (Charles River, Raleigh, NC). Carried out. The same protocol was used as in Example 1, except that the supply of total plasmid DNA was reduced to 50 μg in a volume of 50 μl of PBS. In addition, an irrelevant plasmid DNA (encoding b-galactosidase) injected with ID (using the MDD device) was used as a negative control (n = 4 animals per group). Luciferase activity in the skin was measured as described in Example 1 above.
[0056]
The results shown in FIG. 2 show a very significant gene expression after ID delivery by MDD device. Luciferase activity in the recovered skin site was more than 3000 times greater than the negative control. These results further demonstrate the utility of the method of the invention in delivering gene-based ones in vivo, resulting in high levels of gene expression by the skin.
Embodiment 3
[0057]
ID delivery and expression of model gene therapeutic / prophylactic agents, porcine model
Pigs have long been recognized as a preferred animal model for skin-based delivery studies. Porcine skin is more similar to human skin than rodent skin in full thickness and hair follicle density. Therefore, the pig model (Yorkshire pig, Archer Farms, Belcamp, MD) was used as a tool to predict the utility of this system in humans. The experiments were performed as in Examples 1 and 2 above, except that a different reporter gene system β-galactosidase (Aldevron, Fargo, ND) was used. The total delivered dose was 50 μg in a 50 μl volume. DNA was injected using the following method. Iii) ID delivery by vertical insertion into the skin using a 30 / 31G needle equipped with a device to limit the needle penetration depth to 1.5 mm, i) by the Mantoux method using a 30G needle and a syringe, and iii) ) Injection by ID delivery (MDD device) by vertical insertion into the skin using a 34G needle equipped with a device that limits the needle penetration depth to 1.0 mm. The negative control group consisted of ID delivery of irrelevant plasmid DNA encoding firefly luciferase by i-iii. (Skin sites n = 11 for 4 pigs in ID mantoe group; ID, 30 / 31G, skin sites for 4 pigs on 1.5 mm device n = 11; ID, 34G, 4 pig skin sites on 1 mm device) Skin site n = 10; skin site of four pigs as negative control n = 19) For the negative control, all data from the three ID delivery methods were merged since all three methods gave equivalent results did.
[0058]
Reporter gene activity in the tissues was measured essentially as described in Example 1, except that the b-galactosidase detection assay (Applied Biosystems, Foster City, CA) was used instead of the luciferase assay.
[0059]
The results shown in FIG. 3 indicate strong reporter gene expression in the skin after delivery of all three IDs. While the response in the ID mantoux group was 100 times the background, the ID, 34G, 1 mm (MDD) group increased 300 times above the background, and the ID, 30G, 1.5 mm (30 g, 1.5 mm ) Group increased 20-fold over background. The total reporter gene expression by the skin cells, as measured by the average activity of the reporter gene collected by the excised skin tissue biopsy, was the strongest at 563,523 RLU in the ID, 34G, 1 mm (MDD) group, whereas it was 30G. 200,788 RLU / mg in the mantoux group, 42,470 RLU / mg in the ID (30 g, 1.5 mm) group, and 1,869 RLU / mg in the negative control. Thus, ID delivery by vertical insertion of a 34G, 1.0 mm needle (MDD) is equivalent to standard mantow injection or similar vertical needle insertion and delivery using a longer (1.5 mm), thicker (30 G) needle. In comparison, it results in superior uptake and expression of DNA by skin cells. In a similar test to deliver a visible dye using these three devices and methods, a 34G, 1.0 mm needle provides more consistent delivery to ID tissue than the other two needles / methods. In addition, less "overflow" of the dose to subcutaneous (SC) tissue has been shown.
[0060]
Because all three devices and methods theoretically target the same tissue space, these differences were not expected. However, controlling the depth of delivery using a lateral insertion (Mantoux) method, as compared to a substantially vertical insertion technique achieved by controlling the length of the cannula with a depth limiting hub, Much more difficult. In addition, needle insertion depth and exposed height of the needle outlet are important features associated with reproducible ID delivery without SC "spillover" or leakage on the skin surface.
[0061]
These results further demonstrate the utility of the methods of the present invention for gene-based, in vivo delivery in larger mammals, resulting in high levels of gene expression by skin cells. In addition, the similar skin composition of pigs and humans should provide equivalent clinical improvement in humans.
Embodiment 4
[0062]
Indirect measurement of localized tissue damage after ID delivery
The results presented in Example 3 above suggest that there is an unexpected improvement in the efficacy achieved by ID delivery with MDD compared to that achieved by the Mantoux method with a standard needle. . In addition, the MDD cannula has a shallower insertion depth than a standard needle and does not "kink" into the ID tissue from the side as in a Mantoux injection, so the entire range of mechanically destroyed tissue is limited. Less than. Tissue damage and inflammation result in the release of several inflammatory proteins, chemokines, cytokines and other inflammatory mediators.
[0063]
Thus, the total protein content of the recovered skin site can be used as an indirect measure of tissue damage and localized inflammation caused by the two delivery methods. Total protein levels were measured using the BCA assay (Pierce, Rockford, Ill.) On skin skin biopsies collected from the porcine samples shown in Example 3 above (excluding 30 g, 1.5 mm). As shown in FIG. 4, both delivery methods induced an increase in total protein content compared to untreated skin. However, the total protein concentration in the collected skin biopsy samples in the ID Mantoux group was significantly (p = 0.01, t-test) greater than the corresponding value in the MDD group (2.4 mg / ml vs 1.5 mg). / Ml). These results provide indirect evidence strongly suggesting that mechanical damage to tissue caused by delivery by the methods of the present invention is less than the corresponding damage caused by Mantoux ID injection.
Embodiment 5
[0064]
Induction of an immune response to influenza DNA vaccine after ID delivery in rats
The examples shown above show that a thin gauge microcannula can be used to effectively deliver a model nucleic acid-based compound to the skin, resulting in high levels of gene expression by skin cells. . To examine the utility of delivering a DNA vaccine according to the method of the present invention, rats were immunized with plasmid DNA encoding influenza virus hemagglutinin (HA) from strain A / PR / 8/34 (plasmid was Dr. Harriet Robinson, provided by Emory University School of Medicine, Atlanta, GA). Brown-Norway rats (n = 3 per group) were tested for plasmids in PBS solution by ID using an MDD device or IM into quadriceps using a conventional 30G needle and 1 cc syringe as described in Example 2. Three immunizations (day 0, 21 and 42) with DNA (50 μg per rat delivered by rapid bolus injection in a volume of 50 μl). As a negative control, DNA was applied topically to untreated skin. Serum was collected at 3, 5, 8, and 11 weeks and analyzed by ELISA for the presence of influenza-specific antibodies. Briefly, microtiter wells (Nalge Nunc, Rochester, NY) were coated overnight at 4 ° C. with 0.1 μg of total inactivated virus (A / PR / 8/34, Charles River SPAFAS, North Franklin, CT). . After blocking for 1 hour at 37 ° C. in PBS + 5% skim milk, the plates were incubated for 1 hour at 37 ° C. with serial dilutions of the test sera. The plates are then washed and incubated with horseradish peroxidase-conjugated IgG, H + L chain (Southern Biotech, Birmingham, AL) for an additional 30 minutes at 37 ° C., and then developed with TMB substrate (Sigma, St. Louis, MO). did. Absorbance measurement (A ₄₅₀ ) Was read on a Tecan Sunrise ™ plate reader (Tecan, RTP, NC).
[0065]
The results (FIG. 5) show that delivery of the gene influenza vaccine by the method of the present invention in the absence of added adjuvant induces a strong influenza-specific serum antibody response. The magnitude of this response was comparable to the response induced by IM injection at all time points. There was no detectable response in the local control. Thus, delivery of a genetic vaccine according to the methods of the invention induces an immune response at least as strong as that induced by the conventional IM injection route.
[0066]
To further investigate the delivery of an adjuvanted gene vaccine by the method of the present invention, the plasmid DNA encoding influenza HA described above was prepared using the MPL + TDM Ribi adjuvant system (RIBI Immunochemicals, Hamilton, MT) according to the manufacturer's instructions. did. Rats (n = 3 per group) were immunized and analyzed for influenza-specific serum antibodies as described above. Titers in the ID delivery group were comparable to IM after the first and second immunizations (3-5 weeks, FIG. 6). However, after the third dose, the ID-induced titers were significantly greater than the corresponding titers induced by IM injection (p = 0.03, by t-test) (FIG. 6). At 11 weeks, the average ID induction titer was 42,000, compared to only 4,600 achieved by IM injection. The local control did not show an influenza-specific response. Thus, delivery of a genetic vaccine by the methods of the present invention in the presence of an adjuvant induces a stronger immune response than that induced by the conventional IM injection route.
Embodiment 6
[0067]
Induction of an immune response to influenza DNA / virus "prime-boost" after ID delivery in rats
A recently developed vaccination approach to a number of diseases, including HIV, is the so-called "prime-boost" approach, which uses different vaccine classes in the first "prime" immunization and a second "boost" (Non-Patent Document 15). For example, priming with a plasmid DNA vaccine was followed by a boost with a subunit protein, an inactivated virus or a vectored DNA preparation. To study the delivery of these types of vaccination methods by the method of the present invention, the first experiment of Example 5 was continued for an additional 6 weeks. At week 11, rats primed with DNA were boosted with whole inactivated influenza virus (A / PR / 8/34, 100 μg in 50 μl volume of PBS by rapid bolus injection). Virus was obtained from Charles River SPAFAS (North Franklin, CT). Influenza-specific serum antibody titers were measured at weeks 13 and 17 by ELISA as described above. Both ID delivery and IM injection elicited a strong booster effect (FIG. 7). Mean influenza-specific antibody titers at 17 weeks were comparable for both delivery methods (titer = 540,000) and significantly more than the very weak titers observed after local topical delivery alone (titer = 3200) It was big. Thus, delivery by the methods of the present invention is suitable for a "prime-boost" immunization method, inducing an immune response at least as strong as that induced by the normal IM injection route.
[0068]
The second experiment described in Example 5 was continued for an additional 6 weeks to evaluate the effect of the adjuvant on the "prime-boost" response. At week 11, rats primed with DNA were boosted with whole inactivated influenza virus (A / PR / 8/34, 100 μg in 50 μl volume by rapid bolus injection as described above). Influenza-specific serum antibody titers were measured at weeks 13 and 17 by ELISA as described above. Both ID delivery and IM injection elicited a strong booster effect (FIG. 8). The average titer in the ID delivery group was higher than that by IM injection after virus boosting, and the average ID-MDD (MDD) titer at week 13 was 240,000 for IM injection, 3 for single topical application. , 200 compared to 540,000. Thus, delivery by the methods of the present invention is suitable for a "prime-boost" immunization method including an adjuvant, and induces a stronger immune response than that induced by the normal IM injection route.
Embodiment 7
[0069]
Induction of an immune response to influenza virus vaccine after ID delivery in rats
To examine the utility of conventional vaccine delivery according to the method of the invention, rats were inactivated influenza as described in Example 6 by ID delivery or by intramuscular (IM) injection using a standard needle and syringe. Immunized with the virus preparation. As a negative control, the vaccine solution was applied topically to untreated skin. The high molecular weight of the inactivated influenza virus prevents effective immunization by passive local absorption. As described above, the dose of vaccine was 100 μg total protein in a volume of 50 μl per animal, delivered by rapid bolus injection (within 1 minute). Rats were immunized three times (days 0, 21 and 42) and sera were collected on days 21, 35, 56 and analyzed for influenza-specific antibodies by ELISA as described above. N = 4 rats per group.
[0070]
The results, shown in FIG. 9, indicate that ID delivery induces a strong antigen-specific immune response. Similar levels of antibodies were induced by the two injection routes, IM and ID. The peak geometric mean titer was 147,200 somewhat higher in the ID-MDD (MDD) group compared to 102,400 for IM injection. Topical application of the vaccine only stimulated a very weak response (maximum mean titer = 500). Thus, ID delivery of a high dose of a conventional vaccine induces an immune response at least as strong as that induced by the conventional IM injection route.
Embodiment 8
[0071]
Induction of an immune response to influenza vaccine after ID delivery in pigs
As noted above, pigs have presented an attractive skin model that closely resembles human skin. To test the ID delivery device in vaccine delivery, Yorkshire pigs were immunized with an inactivated influenza vaccine as described above and ID delivery compared to IM injection. Pigs were immunized on days 0, 21 and 49, and sera were collected as described above on days 14, 36, 49 and 60 and analyzed for influenza specific antibodies by ELISA. Pig-specific secondary antibodies were obtained from Bestyl Laboratories (Montgomery, TX).
[0072]
The results (FIG. 10) show that ID delivery induces a strong antigen-specific immune response. Similar levels of antibodies were induced by the two injection routes, IM and ID. The peak geometric mean titer was 1,333, slightly higher in the MDD group compared to 667 with IM injection. Thus, ID delivery of a high dose of a conventional vaccine induces an immune response at least as strong as that induced by the conventional IM injection route.
Embodiment 9
[0073]
ID delivery of low dose influenza vaccine
In Example 7, rats were immunized with 100 μg of inactivated influenza virus by ID injection or IM using a conventional needle and syringe. At such high doses, both delivery methods induced similar levels of serum antibodies, although the ID-induced titers were slightly higher than the IM-induced titers at the last time point.
[0074]
To further test the relationship between the method of delivery and dose level, rats were treated with reduced doses of inactivated influenza virus ranging from 1 μg to 0.01 μg per animal using the ID and IM routes of administration described above. Immunized. Rats were immunized three times (days 0, 21 and 42) and sera were collected on days 21, 35 and 56 and analyzed for serum anti-influenza antibodies (n = 4 rats per group). Similar trends were observed on days 21 and 35, but the data shown in FIG. 11 reflects the titer on day 56. ID delivery (MDD) resulted in a significant antibody response that did not differ significantly in magnitude at the three doses tested. That is, delivery of doses as low as 0.01 μg (10 ng) induced titers comparable to those observed with 100 times more vaccine (1 μg). In contrast, when the IM dose was reduced from 1 μg to 0.1 μg, there was also a significant decrease in titer when the dose was further reduced to 0.01 μg. Moreover, the titer variability induced by ID delivery was significantly lower compared to IM. In particular, no visible side effects (harmful effects on the skin) were observed at the ID administration site.
[0075]
The results strongly indicate that ID delivery by the method of the present invention allows a significant (at least 100-fold) reduction in vaccine dose compared to IM injection. A significant immune response was observed with the nanogram amount of vaccine. Similar benefits would be expected at the clinical stage.
[0076]
The results described herein show that ID injection of vaccines and genetic material can reproducibly achieve the methods of the invention. This method of delivery is easier to perform than standard Mantoux injection or IM, and in one embodiment, is less invasive and painful by limiting and controlling the depth of penetration into the skin. In addition, this method allows for more reproducible ID delivery than a standard needle-based Mantoux injection, resulting in improved targeting of skin cells with the resulting benefits as described above.
[0077]
In addition, the method is compatible with whole inactivated virus vaccines and DNA plasmids, as viral particles or plasmid DNA pass through the microcannula at the relatively high pressures associated with in vivo ID delivery. There is no associated reduction in biological effectiveness as occurs when plasmid DNA is sheared or degraded. The results detailed herein show that a stronger immune response is induced by ID delivery, especially at lower vaccine doses, thus potentially allowing significant dose reductions and combination vaccines in humans. It indicates that you want to.
[0078]
The results shown above demonstrate improved immunization with ID delivery using a device as described above, as compared to standard intramuscular (IM) injection using a regular needle and syringe. Dose reduction studies (Example 9) show that ID delivery induces a serum antibody response to the influenza vaccine in rats with at least 100 times less vaccine than is required for IM injection. If applicable at the clinical stage, such dose reductions will reduce or eliminate the problem of lack of a common influenza vaccine worldwide. Further, the potential for such dose reductions allows for the delivery of a larger number of vaccine antigens in a single dose, thus allowing a combined vaccine. This approach is particularly suitable for HIV vaccines where it is necessary to immunize with several genetic variants / substrains simultaneously to induce protective immunity.
[0079]
Similar benefits are expected for other types of prophylactic and therapeutic vaccines, immunotherapeutics and cell-based ones by improving targeting of the immune system using the methods and devices of the present invention.
[0080]
In other embodiments, ID delivery of the present invention can be accomplished by traditional methods of administration, such as IP, IM, intranasal or other mucosal routes, or SQ injection, to provide improved immunological or therapeutic response. Combination with topical or skin abrasion methods is within the scope of the present invention. This will include the same or different classes of vaccine and / or therapeutic agents, as well as simultaneous or sequential administration.
[0081]
All references cited herein are hereby incorporated by reference. The discussion of the references herein is solely intended to summarize the assertions made by their authors, and is not an admission that any reference constitutes prior art relating to patentability. Applicants reserve the right to review the accuracy and pertinence of the cited references.
[0082]
The embodiments illustrated and discussed herein are intended only to teach those skilled in the art the best ways to make and use the invention known to the inventors and to limit the scope of the invention. It should not be considered limiting. Exemplary embodiments of the present invention may be modified or changed and elements added or omitted without departing from the present invention, as will be appreciated by those skilled in the art in light of the above teachings. it can. Therefore, it is to be understood that, within the scope of the appended claims or their equivalents, the invention may be practiced otherwise than as specifically described.
[Brief description of the drawings]
[0083]
FIG. 1 shows reporter gene activity in guinea pig skin after delivery of firefly luciferase-encoding plasmid DNA. The results are shown as relative light units (RLU) / mg protein for the intradermal delivery by the Mantoux method, the delivery method of the invention, and the control group where plasmid DNA was applied topically to shaved skin.
FIG. 2 shows reporter gene activity in rat skin after delivery of firefly luciferase-encoding plasmid DNA. Results are shown as RLU / mg protein for intradermal delivery by the microdermal delivery method (one embodiment of the present invention, MDD), and controls injected with irrelevant plasmid DNA.
FIG. 3 shows reporter gene activity in porcine skin after delivery of plasmid DNA encoding β-galactosidase. Results are shown as RLU / mg for intradermal delivery by the Mandaux method by ID delivery by MDD device (34 g) to a depth of 1 mm and 1.5 mm, respectively, or by vertical insertion into the skin using a 30 g needle, and for negative controls. Shown as protein.
FIG. 4 shows the Manto ID of reporter plasmid DNA and the total protein content in the recovered skin sites of pigs after MDD delivery. The control ("negative") is untreated skin.
FIG. 5 shows influenza-specific serum antibody responses in rats after delivery of plasmid DNA encoding influenza virus hemagglutinin in the absence of added adjuvant. Plasmid DNA was administered by ID delivery using an MDD device or by intramuscular (IM) injection using a standard needle and syringe. "Topical" indicates a control group to which the preparation was applied topically.
FIG. 6 shows influenza-specific serum antibody responses in rats after delivery of plasmid DNA encoding influenza virus hemagglutinin in the presence of an adjuvant. Plasmid DNA was administered by ID delivery using an MDD device or by intramuscular (IM) injection using a standard needle and syringe. "Topical" indicates a control group to which the preparation was applied topically.
FIG. 7: Influenza-specific serum antibodies in rats after "priming" with plasmid DNA in the absence of added adjuvant, followed by "boost" with whole inactivated influenza virus in the absence of added adjuvant Indicates a response. Plasmid DNA or whole inactivated influenza virus was administered by ID delivery using an MDD device or by intramuscular (IM) injection using a standard needle and syringe. "Topical" indicates a control group to which the preparation was applied topically.
FIG. 8: Influenza-specific serum antibody response in rats after "priming" with plasmid DNA in the presence of added adjuvant, followed by "boost" with whole inactivated influenza virus in the absence of added adjuvant Is shown. Plasmid DNA or whole inactivated influenza virus was administered by ID delivery using an MDD device or by intramuscular (IM) injection using a standard needle and syringe. "Topical" indicates a control group to which the preparation was applied topically.
FIG. 9 shows influenza-specific serum antibody responses in rats to total inactivated influenza virus administered by ID delivery using an MDD device or by intramuscular (IM) injection using a standard needle and syringe. "Topical" indicates a control group to which the preparation was applied topically.
FIG. 10 shows influenza-specific serum antibody responses in pigs to total inactivated influenza virus administered by ID delivery using an MDD device or by intramuscular (IM) injection using a standard needle and syringe.
FIG. 11 shows influenza-specific serum antibody responses in rats to low doses of whole inactivated influenza virus administered by ID delivery using an MDD device or by intramuscular (IM) injection using a standard needle and syringe.

Claims (60)

A method of delivering a vaccine to a mammal, the method comprising intradermally administering the vaccine through at least one hollow needle having an outlet having an exposed height between 0 and 1 mm, wherein the outlet comprises A method of being inserted into the skin at a depth of between 0.5 mm and 2 mm. The vaccine is
A hub portion attachable to a pre-fillable reservoir for storing the vaccine;
At least one hollow microneedle supported by the hub portion and having a forward tip extending away from the hub portion;
An apparatus comprising a limiter portion surrounding said one or more microneedles and extending away from said hub portion toward said front tip of said one or more microneedles, wherein said limiter comprises said one or more microneedles. A generally flat skin-engaging surface extending in a plane generally perpendicular to the axis of the microneedle, adapted to be received against mammalian skin for performing intradermal injection of the vaccine, One or more anterior tips of the plurality of microneedles extend a distance of about 0.5 mm to 2.0 mm beyond the skin engaging surface, and the limiter portion includes a microneedle into the dermis layer of mammalian skin. The method of claim 1, wherein the method is administered with a device that limits the penetration of the drug. 2. The method of claim 1, wherein the delivery of the vaccine occurs at a depth between 0.025 mm and 2.5 mm in the skin of the mammal. 2. The method of claim 1, wherein the delivered vaccine elicits an immune response in a mammal that is equal to or greater than the response following delivery of the same amount of vaccine by subcutaneous or intramuscular injection. 2. The method of claim 1, wherein the delivered vaccine elicits an immune response in a mammal that is equal to or greater than the response following delivery of a larger amount of the vaccine by subcutaneous or intramuscular injection. The method of claim 1, wherein the needle is a microneedle of about 31 to 50 gauge. The method of claim 1, wherein the needle has a tilt angle between 20 and 90 degrees. The method of claim 7, wherein the needle has a tilt angle between 25 and 40 degrees. 7. The method of claim 6, wherein the length of the needle inserted into the skin is from about 0.5 mm to a depth of about 1.7 mm. 7. The method of claim 6, wherein the microneedles are in an array of microneedles. The method of claim 1, wherein the vaccine comprises a live attenuated virus. The method of claim 1, wherein the vaccine comprises live attenuated bacteria. 2. The method of claim 1, wherein the vaccine comprises an inactivated or killed virus. 14. The method according to claim 13, wherein the vaccine further comprises an adjuvant. 14. The method according to claim 13, wherein the vaccine is an influenza vaccine. The method of claim 1, wherein the vaccine comprises inactivated or killed bacteria. The method of claim 1, wherein the vaccine comprises a nucleic acid. 18. The method of claim 17, wherein the peptide or protein encoded by the nucleic acid is expressed in a mammal. 18. The method according to claim 17, wherein the vaccine further comprises an adjuvant. 18. The method according to claim 17, wherein the vaccine is an influenza vaccine. The method of claim 1, wherein the vaccine comprises a live non-attenuated bacterium or virus. The method of claim 1, wherein the vaccine comprises a mammalian cell or a component thereof. 2. The method of claim 1, wherein the vaccine comprises a polysaccharide or polysaccharide complex. The method of claim 1, wherein the vaccine comprises a protein or peptide. The method of claim 1, wherein the one or more needles are inserted substantially perpendicular to the skin. The method of claim 1, further comprising administering the second vaccine intramuscularly, subcutaneously, mucosally, intraperitoneally, intravenously, topically or epidermally. 27. The method of claim 26, wherein the second vaccine is of the same composition as the vaccine. 27. The method of claim 26, wherein the second vaccine has a different composition than the vaccine. 27. The method of claim 26, wherein the second vaccine is a different vaccine class than the vaccine. 27. The method of claim 26, wherein said second vaccine is administered simultaneously with said vaccine. 27. The method of claim 26, wherein the second vaccine is administered sequentially to the vaccine. Further comprising administering the second vaccine via at least one hollow needle having an outlet having an exposed height between 0 and 1 mm, wherein the outlet is at a depth between 0.5 mm and 2 mm. The method of claim 1, wherein the method is inserted into 33. The method of claim 32, wherein delivery of the second vaccine occurs at a depth between 0.025 mm and 2.5 mm in the skin of the mammal. 33. The method of claim 32, wherein a second vaccine is administered concurrently with the vaccine. 33. The method of claim 32, wherein the second vaccine is administered sequentially to the vaccine. 36. The method of claim 35, wherein the second vaccine is administered one day to six weeks after the vaccine. 33. The method of claim 32, wherein the second vaccine is a different vaccine class than the vaccine. 33. The method of claim 32, wherein the second vaccine is of the same composition as the vaccine. 33. The method of claim 32, wherein the second vaccine has a different composition than the vaccine. A method of delivering a vaccine to a mammal, said method comprising:
a) contacting the mammalian skin with a device having dermal access means for accurately targeting the dermal space of the skin with a vaccine at a depth between 0.025 mm and 2.5 mm;
b) delivering the vaccine to the dermal space. 41. The method of claim 40, wherein the vaccine delivered elicits an immune response in a mammal that is equal to or greater than the response following delivery of the same amount of vaccine by subcutaneous or intramuscular injection. 41. The method of claim 40, wherein the delivered vaccine elicits an immune response in a mammal that is equal to or greater than the response following delivery of a larger amount of the vaccine by subcutaneous or intramuscular injection. A method for delivering a gene therapy agent to a mammal, comprising administering the gene therapy agent via at least one small gauge hollow needle having an outlet having an exposed height between 0 and 1 mm. And wherein the outlet is inserted into the skin to a depth between 0.5 mm and 2 mm. 44. The method of claim 43, wherein delivery of the therapeutic agent occurs at a depth between 0.025 mm and 2.5 mm in the skin of the mammal. 44. The method of claim 43, wherein the peptide or protein encoded by the gene therapy agent is expressed in a mammal. 46. The method of claim 45, wherein expression occurs in mammalian skin cells. 44. The method of claim 43, wherein the needle is a 31 to 50 gauge microneedle. 44. The method of claim 43, wherein the needle has a tilt angle between 20 and 90 degrees. 49. The method of claim 48, wherein the needle has a tilt angle between 25 and 40 degrees. 44. The method of claim 43, wherein the needle has a length from about 0.5 mm to about 1.7 mm. 48. The method of claim 47, wherein the microneedles are included in an array of microneedles. 44. The method of claim 43, wherein the gene therapy agent comprises a nucleic acid. 44. The method of claim 43, wherein one or more needles are inserted substantially perpendicular to the skin. A kit comprising a delivery device having at least one hollow needle designed to deliver a substance intradermally to a depth between 0.025 mm and 2.5 mm, wherein the delivery device comprises a gene therapy agent or A kit suitable for receiving a reservoir containing a vaccine, wherein the microneedle is in communication with the reservoir. 55. The kit of claim 54, further comprising an effective dose of a vaccine or gene therapy agent. 56. The kit of claim 55, wherein the dose is contained in a reservoir that is an integral part of the delivery device or in a reservoir that can be operatively attached to the delivery device. 55. The kit of claim 54, wherein the hollow microneedles are about 31 to 50 gauge. 55. The kit of claim 54, wherein the device comprises an array of microneedles. The device
A hub portion attachable to a pre-fillable reservoir for storing the vaccine;
At least one hollow microneedle supported by the hub portion and having a forward tip extending away from the hub portion;
A generally flat skin comprising a limiter portion surrounding the microneedle and extending away from the hub portion toward the front end of the microneedle, wherein the limiter extends in a plane generally perpendicular to the axis of the microneedle. An engaging surface, adapted to be received against mammalian skin for intradermal injection of the vaccine, wherein the microneedle forward tip is about 0.5 mm beyond the skin engaging surface. 55. The kit of claim 54, extending a distance of 2.0 mm, wherein the limiter portion limits penetration of microneedles into the dermis layer of mammalian skin. A kit comprising dermal access means designed to deliver a substance intradermally to a depth between 0.025 mm and 2.5 mm, wherein said dermal access means comprises a gene therapy agent or a vaccine. A kit adapted to receive the dermis, wherein the dermis access means is in communication with the reservoir.

Images